1. Field
Provided are a fusion protein including granzyme B, a cell penetrating peptide, a cleavage site of a peptidase or protease, and a targeting moiety, a pharmaceutical composition for cell membrane penetration including the fusion protein, and an anticancer composition including the fusion protein.
2. Description of the Related Art
It has been generally required in various clinical circumstances to specifically kill a cancer cell. A number of intracellular signal transduction pathways are associated with cell death and cell survival, and thus cell death and cell survival can be controlled by controlling pathways associated with cell death and survival. The pathways can be successfully controlled by the successful delivery of a substance that restricts or blocks the pathway. A representative example of the signal transduction pathways is an apoptosis pathway. Thus, the elements related to the apoptosis pathway are useful as a target to specifically destroy a particular cell.
Granzyme B (GrB), a serine protease, is found in cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Granzyme B relates to apoptosis which is induced in a target cell when it is exposed to the contents of lysosome-type cytoplasmic granules (or lytic granules). Cytotoxic T lymphocyte granules include a series of serine proteases including perforin, a pore-forming protein, and granzymes.
In a lymphocyte-mediated cell lysis, perforin has been known to insert itself into a plasma membrane of a target cell and polymerize, forming a pore, thereby mediating the access of granzyme B into the cytoplasm of the target cell. Once granzyme B is introduced inside of a cell, it directly activates a caspase and induces rapid DNA destruction, thereby inducing cellular apoptosis.
Granzymes are structurally related to each other, but have different substrate preferences. Among the various granzymes, granzyme B has a particular activity to cleave a procaspase after an aspartic acid residue. and causes the maturation (i.e., activation) of the procapsase into caspase-3, thereby exhibiting a high cytotoxicity to a target cell inducing cellular apoptosis. Granzyme B directly activates caspases under certain conditions, directly damaging downstream caspase substrates, to trigger apoptosis.
Recently, various technologies to introduce macromolecules such as proteins into cells have been developed and are central to certain new therapies. However, exact targeting of a cell or tissue is difficult using existing technologies. In an effort to solve this problem, there have been many studies conducted regarding the cell membrane penetration of therapeutic proteins.
It has recently been found that a TAT protein of HIV-1 which is added to a cell culture medium can be delivered inside of a cell, and thus, the protein has been known as a protein transduction domain (PTD). PTD can be transferred into a cell together with other peptide or proteins which are fused thereto. Thus, there have been various attempts to transfer therapeutic drugs, peptides, and proteins into a cell using the PTD.
It is required to develop a technology for intracellular delivery of a cytotoxic substance such as granzyme B using a protein transduction domain.